The long term goals of this research are to determine the structures of mammalian alcohol dehydrogenase (ADH) genes and the mechanism which regulate their expression. Differences in the rate of ethanol metabolism, caused either by differences in the types of ADH isozymes or in the amount of ADH produced, can affect the consequences of alcohol consumption. This renewal continues our focus upon the control of ADH expression, exploiting comparative molecular studies on our cloned human and mouse ADH genes. Gene expression is regulated by the interactions among cis-acting DNA sequences and trans-acting transcriptional factors that bind to them. We will examine both aspects of the regulation of ADH expression. We will locate cis-acting DNA sequences that function in regulation of the human Class I ADH genes and in the related Class I ADH genes from mouse strains that differ in the amount of ADH in liver. The three human genes, ADH1, ADH2, and ADH3, are all expressed in adult liver but are expressed to different extents in non-hepatic tissues. Comparisons among them and quantitative competition experiments will localized sequences responsible for tissue-specific expression. These experiments will use gel retardation and methylation interference assays to determine cis-acting sequences at which transcription factors bind, and functional assays such as in vitro transcription to determine their roles in gene expression. We will clone the region of Chromosome 4 containing the human ADH genes and examine the organization of this gene cluster. Using PCR amplification of portions of the human ADH genes we will search for differences among individuals in the sequences regulating the amount of ADH expressed. We will also clone and sequence the 5' regions of the Class III ch-ADH genes, compare them with the Class I genes, and identify their cis-acting DNA sequences. The data we obtain on cis-acting regulatory sequences in the Class I ADH genes will be used to identify trans-acting protein factors that bind to these sequences and control transcription. These studies will contribute to our understanding of genetic factors underlying differences among individuals in the metabolic and pathological effects of alcohol, and potentially in predisposition to alcohol consumption.